Mixed probiotics capable of degrading fungaltoxin in feed and application

Abstract

The invention discloses mixed probiotics capable of degrading mycotoxin in feed and application and an application method of the mixed probiotics in degrading the mycotoxin in the feed, and the mixed probiotics comprise lactobacillus fermentum, lactobacillus plantarum, saccharomyces cerevisiae, bacillus subtilis and lactobacillus brevis. The mixed probiotics are prepared from the following components in parts by weight: lactobacillus fermentum, lactobacillus plantarum, saccharomyces cerevisiae, bacillus subtilis and lactobacillus brevis in a ratio of 1: (0.2-0.6): (0.1-0.3): (0.2-0.5): (0.2-0.4). According to the application of the mixed probiotics to degradation of the mycotoxin in the feed, the mycotoxin in the feed or feed raw materials can be safely and efficiently inhibited, the fermented feed with low mycotoxin content can be further prepared, and the feed is safe, free of adverse effects such as drug residues and the like, and has a very great application prospect in the feed industry.

Description

Technical Field

[0001] The invention relates to the field of fermented feed, and in particular to a mixed probiotic capable of degrading mycotoxins in feed and an application thereof. Background Art

[0002] Mycotoxins have a wide range of toxic effects on humans and animals, especially causing great harm to animal husbandry production. Mycotoxins are highly toxic secondary metabolites produced by filamentous fungi such as Fusarium, Aspergillus and Penicillium. The occurrence of mycotoxins in feed raw materials is a global problem, especially in grains such as wheat, corn, peanuts, and soybeans. Corn may be contaminated with mycotoxins such as aflatoxin (AFB1), zearalenone (ZEN), and deoxynivalenol (DON) before harvest, during harvest, and during dry storage. AFB1 mainly has hepatotoxic effects and can cause serious damage to the liver. ZEN has estrogenic, genotoxic, immunotoxic, teratogenic, and carcinogenic properties, and DON can cause diarrhea, vomiting, and gastrointestinal inflammation in humans and animals. The high toxicity of mycotoxin contamination can cause huge economic and serious health problems, and the prevention, control, and detoxification of mycotoxins are urgent. At present, the main means of mycotoxin detoxification are physical, chemical, and biological methods, and they have been developed as the main strategies for removing mycotoxins and detoxification. Biological-based methods for degrading toxins have more advantages than physical and chemical methods and are widely considered to be specific, efficient and environmentally friendly. Biological control methods that can prevent and control the growth of toxin-producing fungi and the production of mycotoxins at different stages of the food chain are attracting widespread attention. However, the inhibitory or degradation effect of a single probiotic on mycotoxins is limited. Therefore, it is necessary to study a new mixed probiotic that can improve the degradation effect of mycotoxins by utilizing the mutual compatibility between the components. Summary of the invention

[0003] The purpose of the present invention is to provide a mixed probiotic capable of degrading mycotoxins in feed, and an application and application method thereof for degrading mycotoxins in feed, so as to efficiently and safely degrade mycotoxins in feed or feed raw materials.

[0004] According to the first aspect of the present invention, a mixed probiotic for degrading mycotoxins in feed is provided, wherein the mixed probiotic comprises lactobacillus fermentum, lactobacillus plantarum, saccharomyces cerevisiae, bacillus subtilis, and lactobacillus brevis, wherein each component in the mixed probiotic is lactobacillus fermentum: lactobacillus plantarum: saccharomyces cerevisiae: bacillus subtilis: lactobacillus brevis=1:(0.2-0.6):(0.1-0.3):(0.2-0.5):(0.2-0.4), and the mixed probiotic can degrade mycotoxins in feed. Thus, the lactobacillus fermentum, lactobacillus plantarum, and lactobacillus brevis in the mixed probiotic produce a large amount of organic acids during the fermentation process, reduce the pH value in the fermented feed, and simultaneously inhibit the propagation and fermentation process of the fungi in the early stage of fermentation of the feed or feed raw materials, so as to reduce the formation of mycotoxins metabolized by fungi. The peptidoglycans on the cell wall of saccharomyces cerevisiae can adsorb mycotoxins produced by fungi to reduce the mycotoxin content in the fermented feed. Lactobacillus fermentans, Bacillus subtilis, Saccharomyces cerevisiae, etc. can degrade proteins, polypeptides and cellulose in feed or feed raw materials, produce more small molecule nutrients, and improve the utilization value of feed or feed raw materials. At the same time, the organic acid produced by lactic acid bacteria can promote animal digestion and absorption. Therefore, in this mixed probiotic, due to the compatibility and synergy between the components, the mixed probiotic has the effect of efficiently degrading fungal toxins in feed or feed raw materials. At the same time, since the probiotic itself is safer for animals and there is no problem of drug residue, the mixed probiotic has a very large application prospect in the field of feed.

[0005] In certain embodiments, the viable bacteria count of each component in the mixed probiotics is 3×10 8 -4×10 9 cfu / g, the number of viable Lactobacillus plantarum was 2×10 8 -3×10 9 cfu / g, the number of live bacteria of Saccharomyces cerevisiae is 2-4×10 8 cfu / g, the number of viable Bacillus subtilis is 1-2×10 8 cfu / g, the number of viable Lactobacillus brevis is 1.5-3×10 9 cfu / g.

[0006] In certain embodiments, the fermentation lactobacillus may be any one of the strains numbered 6233, 22808, 6278, and 22808 purchased from CICC (China Industrial Microbiological Culture Collection Center); the plant lactobacillus may be any one of the strains numbered 24936, 22698, 6258, and 25215 purchased from CICC; the saccharomyces cerevisiae may be any one of the strains numbered 32254, 31923, 1355, 1562, and 33674 purchased from CICC; the subtilis may be any one of the strains numbered 20872, 20506, 20825, and 20822 purchased from CICC; the brevis lactobacillus may be any one of the strains numbered 20299, 20297, 20230, and 25206 purchased from CICC, or strains obtained by any means, all of which can achieve the effect of inhibiting / degrading fungal toxins.

[0007] According to a second aspect of the present invention, there is provided an application of a mixed probiotic for degrading mycotoxins in feed, wherein the mixed probiotic comprises Lactobacillus fermentum, Lactobacillus plantarum, Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus brevis, wherein the weight ratio of the components in the mixed probiotic is Lactobacillus fermentum: Lactobacillus plantarum: Saccharomyces cerevisiae: Bacillus subtilis: Lactobacillus brevis = 1: (0.2-0.6): (0.1-0.3): (0.2-0.5): (0.2-0.4); or the number of live bacteria of each component in the mixed probiotic is 3×10 8 -4×10 9 cfu / g, the number of viable Lactobacillus plantarum was 2×10 8 -3×10 9 cfu / g, the number of live bacteria of Saccharomyces cerevisiae is 2-4×10 8 cfu / g, the number of viable Bacillus subtilis is 1-2×10 8 cfu / g, the number of viable Lactobacillus brevis is 1.5-3×10 9 cfu / g. Therefore, through this application, the contamination of three mycotoxins, aflatoxin, zearalenone and deoxynivalenol, on feed or feed raw materials can be efficiently and safely reduced, which has theoretical guiding significance for the degradation of aflatoxin, zearalenone and deoxynivalenol in the modern production of feed.

[0008] In certain embodiments, the fungal toxins include any one or more of aflatoxins, zearalenone, and deoxynivalenol.

[0009] According to a third aspect of the present invention, there is provided a method for degrading mycotoxins in feed by mixing probiotics, wherein the mycotoxins include any one or more of aflatoxin, zearalenone, and deoxynivalenol, and the method comprises the following steps:

[0010] S1: Feed processing: crush the feed, sieve and dry it;

[0011] S2: Preparation of mixed probiotics: Mix the activated and cultured bacterial liquids of fermented Lactobacillus, plantarum Lactobacillus, saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus brevis to prepare a mixed probiotic liquid; the weight ratio of the components in the mixed probiotic liquid is Lactobacillus fermented: Lactobacillus plantarum: saccharomyces cerevisiae: Bacillus subtilis: Lactobacillus brevis = 1: (0.2-0.6): (0.1-0.3): (0.2-0.5): (0.2-0.4); or the number of live bacteria of the components in the mixed probiotic liquid is respectively Lactobacillus fermented: 3×10 8 -4×10 9 cfu / g, the number of viable Lactobacillus plantarum was 2×10 8 -3×10 9 cfu / g, the number of live bacteria of Saccharomyces cerevisiae is 2-4×10 8 cfu / g, the number of viable Bacillus subtilis is 1-2×10 8 cfu / g, the number of viable Lactobacillus brevis is 1.5-3×10 9 cfu / g;

[0012] S3: Degradation of mycotoxins in feed: The mixed probiotics prepared in step S2 are mixed with water to prepare a diluent containing the mixed probiotics, and the mixture is added to the crushed and dried feed to degrade the mycotoxins in the feed. Thus, the method can quickly, conveniently, safely and efficiently degrade the mycotoxins in the feed or feed raw materials.

[0013] In some embodiments, sucrose, calcium carbonate, dipotassium hydrogen phosphate, and sodium acetate are also added to the water used for preparing the diluent containing mixed probiotics in step S3. Adding sucrose, calcium carbonate, dipotassium hydrogen phosphate, and sodium acetate in this method can provide sufficient nutrition for the reproduction of early strains, so as to increase the number of lactic acid bacteria, to improve the growth vitality of lactic acid bacteria, and fully utilized by lactic acid bacteria to produce organic acids; wherein calcium carbonate, dipotassium hydrogen phosphate, and sodium acetate can provide metal ions, can activate nucleic acids and related enzyme activities in bacteria, can also control the permeability of cell membranes, and have a vital role in the reproduction and metabolism of bacterial cells. Thus, by adding these auxiliary materials, the synergistic effect between probiotics can be better exerted, and the effect of mixed probiotics in inhibiting mycotoxin in feed can be further improved.

[0014] According to a fourth aspect of the present invention, there is provided a method for preparing a fermented feed, wherein the fermented feed prepared by the method has a low content of mycotoxins, wherein the mycotoxins include any one or more of aflatoxin, zearalenone, and deoxynivalenol, and the method comprises the following steps:

[0015] S1: Feed raw material processing: The feed raw materials are crushed, sieved and dried.

[0016] S2: Preparation of mixed probiotics: Mix the activated and cultured bacterial liquids of fermented Lactobacillus, plantarum Lactobacillus, saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus brevis to prepare a mixed probiotic liquid; the weight ratio of the components in the mixed probiotic liquid is Lactobacillus fermented: Lactobacillus plantarum: saccharomyces cerevisiae: Bacillus subtilis: Lactobacillus brevis = 1: (0.2-0.6): (0.1-0.3): (0.2-0.5): (0.2-0.4); or the number of live bacteria of the components in the mixed probiotic liquid is respectively Lactobacillus fermented: 3×10 8 -4×10 9 cfu / g, the number of viable Lactobacillus plantarum was 2×10 8 -3×10 9 cfu / g, the number of live bacteria of Saccharomyces cerevisiae is 2-4×10 8 cfu / g, the number of viable Bacillus subtilis is 1-2×10 8 cfu / g, the number of viable Lactobacillus brevis is 1.5-3×10 9 cfu / g; S3: Preparation of fermented feed: The mixed probiotics prepared in step S2 and water are mixed evenly to prepare a dilution containing the mixed probiotics, which is added to the crushed and dried feed raw materials, and then expanded and granulated after anaerobic fermentation to obtain a fermented feed with low mycotoxin content.

[0017] Therefore, by adding mixed probiotics and anaerobic fermentation culture during the feed preparation process, the three mycotoxins, such as aflatoxin, zearalenone, and deoxynivalenol, in the feed can be effectively degraded. In production practice, the contamination of the three toxins, aflatoxin, zearalenone, and deoxynivalenol, on feed or feed raw materials can be effectively reduced, and it has theoretical guiding significance for the degradation of aflatoxin, zearalenone, and deoxynivalenol in the modern production of feed. And a fermented feed with low content of aflatoxin, zearalenone, and deoxynivalenol and safe can be prepared.

[0018] In certain embodiments, the feed raw material in step S1 is one or more of wheat, barley, bran, soybean meal, puffed corn, and corn.

[0019] In certain embodiments, sucrose, calcium carbonate, dipotassium hydrogen phosphate, and sodium acetate are further added to the water used to prepare the diluent containing the mixed probiotics in step S3.

[0020] According to the fifth aspect of the present invention, a fermented feed prepared by the above method is provided. The content of aflatoxin, zearalenone, deoxynivalenol and other mycotoxins in the feed is low, and the feed is a lactic acid bacteria fermented feed, which can also improve the quality of feed, reduce breeding costs, reduce the incidence of disease, promote feeding rate and absorption; promote animal growth and shorten the growth cycle. Feed probiotic additives based on lactic acid bacteria can replace the use of antibiotics, have multiple probiotic effects, improve the safety of feed, and have a very large application prospect in the feed industry.

[0021] Beneficial effects of the present invention:

[0022] 1. The mixed probiotics disclosed in the present invention and its application in the degradation of mycotoxins in feed, wherein fermented lactobacillus, plant lactobacillus and brevis lactobacillus produce a large amount of organic acid during the fermentation process, reduce the pH value in the fermented feed, and inhibit the propagation and fermentation process of the fungi in the early stage of fermentation of feed or feed raw materials, so as to reduce the formation of mycotoxins metabolized by fungi. The peptidoglycans on the cell wall of saccharomyces cerevisiae can adsorb the mycotoxins produced by fungi to reduce the mycotoxin content in the fermented feed. Lactobacillus fermented, bacillus subtilis, saccharomyces cerevisiae, etc. can degrade the protein, polypeptide and cellulose in the feed or feed raw materials, produce more small molecule nutrients, improve the utilization value of the feed or feed raw materials, and the organic acid produced by lactic acid bacteria can promote animal digestion and absorption. Moreover, since the probiotics themselves are safer for animals and do not have the problems of drug residues, the mixed probiotics have a very large application prospect in the field of feed.

[0023] 2. The method for degrading mycotoxins in feed by mixed probiotics disclosed in the present invention can be used to quickly, conveniently, safely and efficiently degrade mycotoxins in feed or feed raw materials. Moreover, by adding sucrose, calcium carbonate, dipotassium hydrogen phosphate and sodium acetate, sufficient nutrition is provided for the reproduction of the early strains, so as to increase the number of lactic acid bacteria, so as to improve the growth vitality of lactic acid bacteria, and fully utilize them to produce organic acids; wherein calcium carbonate, dipotassium hydrogen phosphate and sodium acetate can provide metal ions, can activate nucleic acids and related enzyme activities in bacteria, can also control the permeability of cell membranes, and play a vital role in the reproduction and metabolism of bacterial cells. Thus, by adding these auxiliary materials, the synergistic effect between probiotics can be better exerted, and the effect of mixed probiotics in inhibiting mycotoxins in feed can be further improved.

[0024] 3. The method for preparing fermented feed disclosed in the present invention and the fermented feed prepared therefrom can effectively degrade three mycotoxins, i.e., aflatoxin, zearalenone, and deoxynivalenol, in feed by adding mixed probiotics and anaerobic fermentation culture during the preparation process. In production practice, the contamination of feed or feed raw materials by the three toxins, i.e., aflatoxin, zearalenone, and deoxynivalenol, can be effectively reduced, and the degradation of aflatoxin, zearalenone, and deoxynivalenol in the modern production of feed has theoretical guiding significance. And a fermented feed with low content of aflatoxin, zearalenone, and deoxynivalenol and safety can be prepared. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the experimental design for the degradation of mycotoxins in corn fermented feed using mixed probiotics. DETAILED DESCRIPTION

[0026] The present invention is described in detail by the following specific embodiments, but it should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps, and conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods and techniques well known to those skilled in the art, and the bacterial agents and test materials used are all obtained through commercial channels.

[0027] YPD medium (yeast extract peptone glucose medium): 10 g of yeast extract, 20 g of peptone, add 900 mL of water, 100 mL of 20% glucose solution, sterilize at 121° C. for 15 min, and then mix in a sterile environment.

[0028] LB medium (Luria-Bertani medium): 5 g beef extract, 10 g peptone, 5 g sodium chloride, dilute to 1000 mL with water, and sterilize at 121°C for 15 min.

[0029] MRS medium (lactic acid bacteria medium): peptone 10g, beef extract 10g, yeast extract 10g, sodium acetate 5g, diammonium citrate 2g, Tween 80 1g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, manganese sulfate 0.2g, pH 6.2-6.4, add 900mL water, 20% glucose solution 100mL, sterilize at 121°C for 15min and then mix under sterile environment.

[0030] Detection methods for three fungal toxins [aflatoxin (AFB1), zearalenone (ZEN), and deoxynivalenol (DON)]: AFB1 is detected by high performance liquid chromatography according to the method in GB / T 36858-2018, ZEN is detected by high performance liquid chromatography according to the method in GB 5009.209-2016, and DON is detected by high performance liquid chromatography according to the method in GB / T30956-2014.

[0031] Saccharomyces cerevisiae (purchased from CICC, No. 33674) was inoculated into YPD medium at 2% and cultured at 37°C; Bacillus subtilis (purchased from CICC, No. 20872) was inoculated into LB medium at 2% and cultured at 37°C; Lactobacillus fermentum (purchased from CICC, No. 22808), Lactobacillus plantarum (purchased from CICC, No. 22698), and Lactobacillus brevis (purchased from CICC, No. 20299) were inoculated into MRS medium at 2% and cultured at 37°C respectively; the culture time was such that the concentration of each bacterial solution could reach 10 9 cfu / mL. The cultured bacterial solution was used as a single bacterial species or two bacterial agents, and the degradation of mycotoxins by the bacterial agents was compared according to the ratio (weight parts) in Table 1. The total number of viable bacteria contained in each gram of the bacterial agent was 10 9 CFU.

[0032] Table 1 Probiotic test groups

[0033] A B C D E F G H Lactobacillus fermentum CICC22808 1 1 Lactobacillus plantarum CICC22698 1 1 Saccharomyces cerevisiae CICC33674 1 1 1 Bacillus subtilis CICC20872 1 1 1 Lactobacillus brevis CICC20299 1

[0034] The fermented lactobacillus may also be any one of strains numbered 6233, 22808, 6278, and 22808 purchased from CICC; the plant lactobacillus may also be any one of strains numbered 24936, 22698, 6258, and 25215 purchased from CICC; the saccharomyces cerevisiae may also be any one of strains numbered 32254, 31923, 1355, 1562, and 33674 purchased from CICC; the subtilis may also be any one of strains numbered 20872, 20506, 20825, and 20822 purchased from CICC; the breve lactobacillus may also be any one of strains numbered 20299, 20297, 20230, and 25206 purchased from CICC, or strains obtained by any method, all of which can achieve the above-mentioned effect of inhibiting / degrading fungal toxins.

[0035] Example 1: Degradation of mycotoxins by mixed probiotics

[0036] The bacterial preparations were prepared according to the ratios of the probiotics in Table 1. The obtained bacteria were resuspended in 1 mL of 1 mg / L AFB1, ZEN, and DON working solutions, and the bacterial concentration was adjusted to 10 9 cfu / mL, placed in a shaker at 150 rpm and cultured at 37°C for 24 hours. Centrifuge the culture solution at 12000 rpm, 4°C for 10 minutes, take the supernatant and filter it with a 0.22μm filter head, and then use HPLC to detect the remaining concentration of fungal toxins. At the same time, set the toxin solution of the same concentration without adding bacteria as a negative control. Among them, toxin degradation rate = (initial toxin concentration - toxin concentration after treatment) / initial toxin concentration × 100%.

[0037] Table 2 Probiotic toxin degradation rate

[0038] serial number AFB1 degradation rate (%) ZEN degradation rate (%) DON degradation rate (%) A 45.53 42.87 52.87 B 41.41 46.27 47.50 C 57.96 53.95 32.87 D 28.82 28.20 46.07 E 24.64 27.11 42.73 F 36.29 30.57 25.47 G 31.93 33.19 25.43 H 27.53 29.87 41.87

[0039] As shown in Table 2, Saccharomyces cerevisiae has a strong ability to degrade aflatoxin AFB1, Bacillus subtilis has a strong ability to degrade zearalenone ZEN, and the three lactic acid bacteria can show a good ability to degrade deoxynivalenol DON. After the probiotics are mixed, the ability to degrade the three fungal toxins has a synergistic effect, which can effectively improve the ability to degrade fungal toxins.

[0040] Example 2: Degradation of mycotoxins by mixed probiotics

[0041] The mycotoxin degradation effect was determined according to the method of Example 1, except that lactic acid bacteria were added to the cultured bacterial solution as a composite bacterial agent based on the same ratio (weight parts) of saccharomyces cerevisiae and Bacillus subtilis, and the mixed probiotic agent was prepared according to the ratio of the bacterial agent in Table 3. The total number of viable bacteria contained in each gram of the bacterial agent was 10 9 CFU. The bacterial agents were prepared and grouped for determination according to Table 3, and the results are shown in Tables 4 and 5.

[0042] Table 3 Mixed probiotics compound table

[0043] serial number Lactobacillus fermentum Lactobacillus plantarum Lactobacillus brevis 1 1 1 1 2 1 2 3 3 1 3 2 4 2 1 3 5 2 2 2 6 2 3 1 7 3 1 2 8 3 2 1 9 3 3 3

[0044] Table 4 Toxin degradation rate of mixed probiotics

[0045] serial number AFB1 degradation rate (%) ZEN degradation rate (%) DON degradation rate (%) 1 <![CDATA[65.92 d ]]> <![CDATA[65.52 d ]]> <![CDATA[62.00 ef ]]> 2 <![CDATA[66.66 d ]]> <![CDATA[67.94 d ]]> <![CDATA[65.30 e ]]> 3 <![CDATA[64.25 d ]]> <![CDATA[66.22 d ]]> <![CDATA[66.07 e ]]> 4 <![CDATA[71.73 c ]]> <![CDATA[69.60 cd ]]> <![CDATA[69.07 d ]]> 5 <![CDATA[73.16 bc ]]> <![CDATA[72.00 c ]]> <![CDATA[69.67 d ]]> 6 <![CDATA[71.07 c ]]> <![CDATA[77.55 b ]]> <![CDATA[71.50 c ]]> 7 <![CDATA[75.85 b ]]> <![CDATA[78.95 b ]]> <![CDATA[68.37 d ]]> 8 <![CDATA[81.59 a ]]> <![CDATA[82.67 a ]]> <![CDATA[79.98 a ]]> 9 <![CDATA[76.53 b ]]> <![CDATA[78.87 b ]]> <![CDATA[75.87 b ]]>

[0046] Table 5 Variance analysis table

[0047]

[0048] a:R 2 =0.958

[0049] b:R2 =0.856

[0050] c:R 2 =0.701

[0051] It can be seen from the results in Tables 4 and 5 that the degradation rate of the three toxins is best when the weight ratio of lactic acid bacteria added to the mixture is Lactobacillus fermentation: Lactobacillus plantarum: Lactobacillus brevis = 3:2:1 based on Saccharomyces cerevisiae and Bacillus subtilis (weight ratio of 1:1), and the weight ratio of each component in the mixed bacteria is Lactobacillus fermentation: Lactobacillus plantarum: Saccharomyces cerevisiae: Bacillus subtilis: Lactobacillus brevis = 3:2:1:1:1.

[0052] Example 3: Degradation of mycotoxins in corn fermented feed by mixed probiotics

[0053] The schematic diagram of the experimental design scheme for the degradation of mycotoxins in corn fermented feed (the main raw material components of the feed are corn: wheat: puffed corn: soybean meal in a ratio of 6:3:3:1) by mixed probiotics is shown in the figure. Figure 1 As shown: first, the feed raw materials are processed and prepared; then the mixed probiotics are prepared, and finally the mixed bacterial liquid is inoculated into the feed for fermentation.

[0054] Step 1: Feed raw material processing: crush the feed raw materials, sieve and dry them.

[0055] Step 2: Preparation of mixed probiotics: according to the preparation method of Example 1, the activated cultured Lactobacillus fermentation, Lactobacillus plantarum, Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus brevis are mixed to obtain a mixed probiotic solution. The weight ratio of each bacterial solution in the mixed bacterial solution is: Lactobacillus fermentation: Lactobacillus plantarum: Saccharomyces cerevisiae: Bacillus subtilis: Lactobacillus brevis = 1: (0.2-0.6): (0.1-0.3): (0.2-0.5): (0.2-0.4).

[0056] The number of viable bacteria in each bacterial solution was 3×10 8 -4×10 9 cfu / g, the number of viable Lactobacillus plantarum was 2×10 8 -3×10 9 cfu / g, the number of live bacteria of Saccharomyces cerevisiae is 2-4×10 8 cfu / g, the number of viable Bacillus subtilis is 1-2×10 8 cfu / g, the number of viable Lactobacillus brevis is 1.5-3×10 9 cfu / g.

[0057] Step 3: Preparation of fermented feed and degradation of mycotoxins in feed: The mixed probiotic solution prepared in step 2 is mixed with water, wherein the water fully dissolves sucrose, calcium carbonate, dipotassium hydrogen phosphate, and sodium acetate, and then the mixed probiotic solution is added to prepare a diluent containing mixed probiotics. Then the diluent containing mixed probiotics is added to the crushed and dried feed raw materials, placed in a closed container, the fermentation raw materials occupy 2 / 3 of the closed container, the fermentation temperature is 37°C, the culture time is 7d, and after anaerobic fermentation, it is expanded and granulated to obtain fermented feed, and the mycotoxins in the fermented feed can be effectively degraded by the mixed probiotics.

[0058] Step 4: Detection of mycotoxin degradation in fermented feed: Take the fermented feed, dilute it appropriately with distilled water, shake it thoroughly, centrifuge it at 4°C for 10 minutes, take the supernatant and filter it with a 0.22μm filter head, and then use HPLC to detect the mycotoxin concentration in the fermented feed. The toxin concentration at the start of fermentation of the fermented feed is taken as the initial concentration, and the toxin degradation rate is calculated as follows: toxin degradation rate = (initial toxin concentration at the start of fermentation - toxin concentration after fermentation) / initial toxin concentration at the start of fermentation × 100%. The results are shown in Table 6:

[0059] Table 6 Degradation of mycotoxins after fermentation

[0060] AFB1 ZEN DON Toxin degradation rate (%) 52.76 49.55 48.48

[0061] As shown in Table 6, corn and soybean meal can effectively degrade three fungal toxins, namely aflatoxin AFB1, zearalenone ZEN and deoxynivalenol DON, after fermentation with probiotics: aflatoxin AFB1 can be degraded by 52.76%, zearalenone ZEN can be degraded by 49.55%, and deoxynivalenol DON can be degraded by 48.48%.

[0062] In a closed container, the fermented feed material occupies 2 / 3 of the space, and the remaining part is air. Saccharomyces cerevisiae quickly uses the oxygen in the container to grow and reproduce, providing favorable environmental conditions for the growth and reproduction of lactic acid bacteria and Bacillus subtilis. At the same time, it can also secrete proteases and cellulases to degrade and utilize the fermentation raw materials. Due to the anaerobic conditions formed, lactic acid bacteria and Bacillus subtilis undergo anaerobic fermentation, and the organic acids they produce reduce the pH value of the feed, thereby inhibiting the growth and reproduction of molds and the formation of mycotoxins during the fermentation process. At the same time, a large amount of peptidoglycan on the surface of probiotics can adsorb mycotoxins and reduce the content of mycotoxins. Therefore, under the synergistic effect of the compatibility of multiple bacteria, the production and content of mycotoxins are inhibited, and the quality of the feed is improved.

[0063] The above-mentioned embodiments are descriptions of the technical solutions, which are only some embodiments of the present invention, not all. The common sense such as the known specific characteristics of the solutions is not described in detail here. For those skilled in the art, based on the structural system of the present invention, several modifications and changes can be made, which should also fall within the scope of protection of the claims of the present invention.

Claims

1. A mixed probiotic capable of degrading mycotoxins in feed, wherein: The mixed probiotics include lactobacillus fermentation, lactobacillus plantarum, saccharomyces cerevisiae, bacillus subtilis and lactobacillus brevis. The weight ratio of the components in the mixed probiotics is lactobacillus fermentation: lactobacillus plantarum: saccharomyces cerevisiae: bacillus subtilis: lactobacillus brevis=1:(0.2-0.6):(0.1-0.3):(0.2-0.5):(0.2-0.4). The mixed probiotics can degrade fungal toxins in feed.

2. The mixed probiotic according to claim 1, wherein: The number of live bacteria of each component in the mixed probiotics is 3×10 8 -4×10 9 cfu / g, the number of viable Lactobacillus plantarum was 2×10 8 -3×10 9 cfu / g, the number of live bacteria of Saccharomyces cerevisiae is 2-4×10 8 cfu / g, the number of viable Bacillus subtilis is 1-2×10 8 cfu / g, the number of viable Lactobacillus brevis is 1.5-3×10 9 cfu / g.

3. Use of the mixed probiotics described in claim 1 or 2 in degrading mycotoxins in feed.

4. The use according to claim 3, wherein: The fungal toxins include any one or more of aflatoxins, zearalenone, and deoxynivalenol.

5. A method for degrading mycotoxins in feed using the mixed probiotics as claimed in claim 1 or 2, wherein: The fungal toxins include any one or more of aflatoxin, zearalenone, and deoxynivalenol, and the method includes the following steps: S1: Feed processing: The feed is crushed, sieved and dried. S2: Preparation of mixed probiotics: Mixing the activated cultured bacterial liquids of fermented Lactobacillus, plantarum Lactobacillus, saccharomyces cerevisiae, bacillus subtilis and lactobacillus brevis to prepare a mixed probiotic liquid; the weight ratio of the components in the mixed probiotic liquid is: fermented Lactobacillus: plantarum Lactobacillus: saccharomyces cerevisiae: bacillus subtilis: lactobacillus brevis = 1: (0.2-0.6): (0.1-0.3): (0.2-0.5): (0.2-0.4); S3: Degradation of mycotoxins in feed: The mixed probiotics prepared in step S2 are mixed with water to prepare a dilution containing the mixed probiotics, which is added to the crushed and dried feed to degrade the mycotoxins in the feed.

6. The method according to claim 5, wherein: In the step S3, sucrose, calcium carbonate, dipotassium hydrogen phosphate and sodium acetate are added to the water used for preparing the diluent containing the mixed probiotics.

7. A method for preparing a fermented feed, wherein: The fermented feed prepared by the method has a low content of mycotoxins, wherein the mycotoxins include any one or more of aflatoxin, zearalenone, and deoxynivalenol. The method comprises the following steps: S1: Feed raw material processing: crushing, sieving and drying the feed raw materials; S2: Preparation of mixed probiotics: Mix the activated and cultured bacterial liquids of fermented Lactobacillus, plantarum Lactobacillus, saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus brevis to prepare a mixed probiotic liquid; the weight ratio of the components in the mixed probiotic liquid is Lactobacillus fermented: Lactobacillus plantarum: saccharomyces cerevisiae: Bacillus subtilis: Lactobacillus brevis = 1: (0.2-0.6): (0.1-0.3): (0.2-0.5): (0.2-0.4) or the number of live bacteria of the components in the mixed probiotic liquid is respectively Lactobacillus fermented: 3×10 8 -4×10 9 cfu / g, the number of viable Lactobacillus plantarum was 2×10 8 -3×10 9 cfu / g, the number of live bacteria of Saccharomyces cerevisiae is 2-4×10 8 cfu / g, the number of viable Bacillus subtilis is 1-2×10 8 cfu / g, the number of viable Lactobacillus brevis is 1.5-3×10 9 cfu / g; S3: Preparation of fermented feed: The mixed probiotics prepared in step S2 and water are evenly mixed to prepare a dilution containing the mixed probiotics, which is added to the crushed and dried feed raw materials, and then expanded and granulated after anaerobic fermentation to obtain a fermented feed with low mycotoxin content.

8. The method according to claim 7, wherein: The feed raw material in step S1 is one or more of wheat, barley, bran, soybean meal, puffed corn, and corn.

9. The method according to claim 7, wherein: In the step S3, sucrose, calcium carbonate, dipotassium hydrogen phosphate and sodium acetate are added to the water used for preparing the diluent containing the mixed probiotics.

10. Fermented feed prepared by the method according to claim 8 or 9.

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